Motoneuron neurodegenerative diseases present major public health issues. For example, amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Approximately 20% of familial ALS is linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene (Julien, J. P., Cell (2001) 104:581–591). Transgenic mice overexpressing this mutant gene (mSOD1G93A) develop a dominantly inherited adult-onset paralytic disorder that has many of the clinical and pathological features of familial ALS (Gurney et al., Science (1994) 264:1772–1775). However, to date, the molecular mechanisms leading to motoneuron degeneration in ALS and most motor neuron diseases remain poorly understood. Because the mechanism leading to motoneuron degeneration in ALS is not known, there is currently no therapy available to prevent or cure ALS.
Glial cell line-derived neurotrophic factor (GDNF) has been shown to be the most potent neurotrophic factor for the proliferation, differentiation, and survival of spinal motoneurons. GDNF and GDNF mRNA levels have been reported to be up-regulated in denervated muscles as found in ALS, polymyostits (PM) and muscular dystrophy (MD), or after peripheral nerve lesion and the like (Trupp et al., J. Cell Biol. (1995) 130:137–148; Lie and Weis, Neurosci. Lett. (1998) 250: 87–90; Yamamoto et al., Neurochem. Res. (1999) 24:785–790). GDNF has been proposed as a therapeutic agent to treat motor neuron disease (Henderson et al., Science (1994) 266:1062–1064; Oppenheim et al., Nature (1995) 373:344–346; Yan et al., Nature (1995) 373:341–344; Sagot et al., J. Neurosci. (1996) 16: 2335–2341; Bohn, M. C., Biochem. Pharmacol. (1999) 57:135–142; Mohajeri et al., Hum. Gene Ther. (1999) 10:1853–1866). Neurotrophic factors such as GDNF have been shown to slow motoneuron degeneration and to restore the function of non-functional motoneurons that are still alive (Trupp et al., J. Cell Biol. (1995) 130:137–148; Sagot et al., J. Neurosci. (1998) 18: 1132–1141; Lie and Weis, Neurosci. Lett. (1998) 250: 87–90; Baumgartner and Shine, J. Neurosci. Res. (1998) 54: 766–777; Yamamoto et al., Neurochem. Res. (1999) 24:785–790; Biesch and Tuszynski, J. Comp. Neurol. (2001) 436:399–410; Keller-Peck et al., J. Neurosci. (2001) 21:6136–6146.
To date, however, clinical trials using repeated administration of recombinant GDNF, as well as other neurotrophic factors such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-I (IGF-I), have shown limited or no promise and/or have resulted in severe side-effects (Yuen, E. C., Phys. Med. Rehabil. Clin. N. Am. (2001) 12:293–306). In particular, these proteins have a short in vivo plasma half-life, have poor access to spinal cord motoneurons, and cause inflammatory reactions that prevent administration at an adequate dose (Haase et al., Nat. Med. (1997) 3:429–436; Alisky and Davidson, Hum. Gene Ther. (2000) 11:2315–2329). These limitations, together with the chronicity and progressive nature of most motoneuron degenerative diseases, underscore the necessity to develop innovative strategies that offer more effective and long-term delivery of neurotrophic factors to motoneurons. In an attempt to overcome the above-described problems, experimenters have studied gene-therapy approaches for treating ALS (Alisky and Davidson, Hum. Gene Ther. (2000) 11:2315–2329). Genetically modified myoblast-based GDNF gene delivery in muscles prevented loss of spinal motoneurons and delayed the onset of the disease in a transgenic mouse familial ALS model (Mohajeri et al., Hum. Gene Ther. (1999) 10:1853–1866). However, the level of GDNF protein in treated muscle was undetectable.
Adeno-associated virus (AAV) has shown promise for delivering genes for gene therapy in clinical trials in humans (see, e.g., Kay et al., Nat. Genet. (2000) 24:257–261). As the only viral vector system based on a nonpathogenic and replication-defective virus, recombinant AAV virions have been successfully used to establish efficient and sustained gene transfer of both proliferating and terminally differentiated cells in a variety of tissues (Bueler, H., Biol. Chem. (1999) 380:613–622). Notwithstanding these successes, AAV-mediated GDNF gene therapy for treating motor neuron disease, such as ALS, has not been demonstrated.
The AAV genome is a linear, single-stranded DNA molecule containing about 4681 nucleotides. The AAV genome generally comprises an internal nonrepeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including as origins of DNA replication, and as packaging signals for the viral genome. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package into a virion. In particular, a family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3.
AAV has been engineered to deliver genes of interest by deleting the internal nonrepeating portion of the AAV genome (i.e., the rep and cap genes) and inserting a heterologous gene between the ITRs. The heterologous gene is typically functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.
AAV is a helper-dependent virus; that is, it requires coinfection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia), in order to form AAV virions. In the absence of coinfection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to replicate and package its genome into an infectious AAV virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells coinfected with a canine adenovirus.
However, prior to the present invention, AAV-mediated delivery of GDNF for the treatment of motoneuron diseases, such as ALS, has not been successfully achieved.